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1.
Sci Rep ; 10(1): 16082, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999417

RESUMO

High-protein diets contribute to an increase in urea follicular concentrations associated with decreased fertility. Urea has been shown to interfere with the epidermal growth factor (EGF)/EGFR system, which has been shown to have a beneficial effect during in vitro maturation (IVM) of oocytes. Of note, the number of cumulus-oocyte complexes (COCs) in the maturation medium can change the maturation and the developmental competence of COCs. Therefore, it was hypothesized that, the presence of urea and EGF may have a differential effect on the depletion/appearance of AAs and competence of COCs matured individually (I-IVM system) or in groups (G-IVM system). In the G-IVM system, COCs increased consumption (depletion) of AAs compared with other groups in the presence of high-level urea (40 mg/dl) + EGF (10 ng/ml). In the I-IVM system, the non-cleaved COCs depleted more AAs than the cleaved COCs, in particular in the presence of urea. The combination of urea and EGF increased the depletion of AAs in the G-IVM system. However, the EGF abrogated the urea-induced depletion of AAs by the I-IVM COCs. The use of N-acetyl-L-cysteine as an EGFR inhibitor canceled urea-induced depletion of AAs. This shows the inhibiting effect of urea over the EGF/EGFR system. In the presence of urea + EGF, COCs had a lower degree of developmental competence than control in both I- and G-IVM systems. Arginine had the best predictive power to identify highly competent COCs in the G-IVM system, while glutamine was the best predictor of the cleavage in the I-IVM system. In conclusion, this multi-level study shows that COCs matured individually or in groups may have different association with AAs metabolism. These findings provide new insights into the relationships between AA metabolism and the subsequent developmental competence of COCs.


Assuntos
Aminoácidos/metabolismo , Oócitos/metabolismo , Aminoácidos Essenciais/metabolismo , Animais , Bovinos , Meios de Cultura , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas In Vitro , Simulação de Acoplamento Molecular , Análise Multinível , Oócitos/citologia , Oócitos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ureia/metabolismo , Ureia/farmacologia
2.
PLoS One ; 15(9): e0239151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941516

RESUMO

The estrogen-signalling pathway is critical for normal follicular development; however, little is known about its importance during in vitro maturation (IVM) in large animals, particularly yaks (Bos grunniens). Through the present study, we aimed to determine the mechanisms underlying estrogen involvement in cumulus expansion and the subsequent development of cumulus-oocyte complexes (COCs). COCs were cultured in the maturation medium supplemented with different concentrations (10-6-10-3 mM) of 17ß-estradiol (E2) or its receptor antagonist, fulvestrant, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot were performed to determine the expression of cumulus-expansion related factors and oocyte-secreted factors (OSFs). The cumulus expansion of COCs was observed using an inverted microscope, and COCs developmental ability were judged by the evaluation of cleavage and blastulation rates per inseminated oocytes by IVF, and the number of cells in the blastocyst. Cumulus expansion increased with 10-6-10-3 mM E2, but decreased with fulvestrant. HAS2, PTGS2, PTX3 and OSFs expression increased in the 10-6-10-3 mM E2 groups. Significantly higher cleavage and blastocyst rates were observed in the 10-4 mM E2 group than in the fulvestrant and 0 mM E2 groups. Moreover, in the 10-4 mM group, blastocysts at 7 days had higher cell counts than the other groups. In conclusion, the increase in cumulus expansion and subsequent oocyte development after the addition of E2 to IVM medium may have resulted from increased cumulus-expansion-related factor expression and OSF levels.


Assuntos
Bovinos/fisiologia , Células do Cúmulo/efeitos dos fármacos , Estrogênios/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Animais , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Feminino , Oócitos/citologia , Oócitos/metabolismo
3.
PLoS One ; 15(9): e0238812, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915922

RESUMO

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.


Assuntos
Bioimpressão , Células do Cúmulo/citologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/citologia , Animais , Automação , Cápsulas , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ovinos
4.
PLoS One ; 15(3): e0229043, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182244

RESUMO

Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.


Assuntos
Meios de Cultura/farmacologia , Células do Cúmulo/citologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/citologia , Anfirregulina/farmacologia , Animais , Biomarcadores , Células Cultivadas , Meios de Cultura/química , Células do Cúmulo/metabolismo , Dinoprostona/farmacologia , Feminino , Fertilização In Vitro , Fluoresceínas/metabolismo , Meiose , Modelos Animais , Peptídeo Natriurético Tipo C/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano/efeitos dos fármacos , Ovinos
5.
Front Biosci (Schol Ed) ; 12: 116-136, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32114451

RESUMO

Oocyte quality influences early embryonic survival, establishment and maintenance of pregnancy, fetal development and adult diseases. The developmental competence of oocytes is acquired gradually and increases with follicular development. The ability of an oocyte to develop into an embryo depends on, having enough specific information in the form of mRNA or proteins. If this information is insufficient, defects in nuclear or cytoplasmic maturation, or in both processes, may arise and thus affect the in vitro development of fertilized oocytes. The greater developmental competence of oocytes aspirated from larger follicles is reported as compared with smaller follicles. Oocyte developmental competence is greatly correlated with the morphology of the cumulus oocyte complexes (COCs). Apart from morphological or biochemical markers, molecular markers have also been investigated. Until now, no specific markers of oocyte developmental competence could be described for the oocyte developmental competence. To, utilize female germplasm to its maximum, there is a need to enhance developmental competence of lesser competent oocytes derived from the follicles which are not fully grown. The oocyte pre-maturation and maturation conditions affect gene expression not only in the oocyte but till the blastocyst stages too. Strategies have been discussed in this review would be useful to enhance the developmental competence of oocytes.


Assuntos
Oócitos/fisiologia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Células do Cúmulo/citologia , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Humanos , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oogênese/fisiologia , Gravidez
6.
Sci Rep ; 10(1): 3493, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103136

RESUMO

Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate and kinetics of glucose consumption and glycolysis throughout IVM. Approximately 95% of glucose consumed was accounted for by lactate production; however, high concurrent oxygen consumption indicated a comparatively increased role for non-glycolytic oxidative phosphorylation. Up to 38% of equine COC oxygen consumption could be attributed to non-mitochondrial activities and there was a significant loss of spare respiratory capacity over the course of IVM. Notably, our data also revealed that current IVM protocols may be failing to satisfy the metabolic demands of the equine COC. Our findings constitute the first report on mitochondrial efficiency in the equine COC and provide new insight into comparative gamete biology as well as metabolism of the COC during in vitro maturation.


Assuntos
Metabolismo Energético , Oócitos/metabolismo , Animais , Células Cultivadas , Células do Cúmulo/citologia , Glucose/metabolismo , Glicólise , Cavalos , Técnicas de Maturação in Vitro de Oócitos , Mitocôndrias/metabolismo , Oócitos/citologia , Consumo de Oxigênio
7.
Cryobiology ; 92: 161-167, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917962

RESUMO

The objective was to evaluate the developmental competence of immature and matured ovine oocytes after removing, maintaining or adding cumulus cells (CC) associated to vitrification by Cryotop method. Three experiments were performed involving 3,144 oocytes. In Experiment 1, CC were removed from immature, matured or fertilized oocytes subjected to in vitro embryo production. In Experiment 2, oocytes were vitrified either in MI or MII stage with or without CC, while a control group with CC remained unvitrified. In Experiment 3, oocytes partially denuded from CC were vitrified either in MI or MII stage, and a co-culture of fresh CC was added or not soon after warming to complete in vitro maturation (IVM) and in vitro fertilization (IVF), or IVF, respectively, while a control group remained unvitrified. In Experiment 1, the cleavage rate, development rate on Day 6 and blastocyst rate on Day 8 were improved when CC were maintained until the end of IVF (P < 0.05). In Experiment 2, vitrification of oocytes with enclosed CC showed a tendency to increase cleavage (P = 0.06) and improved blastocyst rate (P < 0.05). In Experiment 3, adding CC as co-culture after vitrification-warming tended to improve cleavage rate (P = 0.06) and increased hatching rate (P < 0.05). Regarding oocyte stage, vitrification of in vitro matured oocytes resulted in greater developmental competence than immature stages (P < 0.05). In conclusion, CC seems to have a relevant role for in vitro embryo development in either fresh or vitrified oocytes.


Assuntos
Criopreservação/métodos , Células do Cúmulo/citologia , Desenvolvimento Embrionário/fisiologia , Oócitos/citologia , Oogênese/fisiologia , Vitrificação , Animais , Blastocisto/citologia , Técnicas de Cocultura , Feminino , Fertilização In Vitro/métodos , Técnicas de Maturação in Vitro de Oócitos , Ovinos
8.
Mol Cell Endocrinol ; 499: 110591, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31546019

RESUMO

Metformin (MET) is the most widely prescribed hypoglycemic drug in type 2 diabetes and Polycystic Ovary Syndrome. Besides its effects on glucose metabolism, MET exerts beneficial effects on these patients' fertility. However, the exact mechanisms of action of MET on female fertility are still unclear. In this work, we analyzed a possible direct effect of MET on ovarian cells. We found expression of the organic cation transporters OCT1, OCT2 and OCT3, responsible for MET uptake into the cells, in rat granulosa cells and human cumulus cells. Furthermore, MET increased pAMPK and decreased VEGF levels both in vivo and in rat granulosa cells in culture. These last effects were reversed when OCTs were inhibited. Our results suggest that MET acts directly on ovarian cells regulating cell metabolism and VEGF expression. Our findings are relevant to optimize PCOS fertility treatment and to explore ovarian MET actions in other female pathologies.


Assuntos
Adenilato Quinase/metabolismo , Células do Cúmulo/citologia , Metformina/administração & dosagem , Fatores de Transcrição de Octâmero/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Animais , Proliferação de Células/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metformina/farmacologia , Modelos Animais , Fosforilação/efeitos dos fármacos , Ratos
9.
Theriogenology ; 142: 320-327, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711691

RESUMO

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ±â€¯1.2 vs. 100% ±â€¯0.0), presence of first polar body (65.9% ±â€¯1.2 vs. 70.5% ±â€¯1.8), nuclear status in second metaphase (62.5% ±â€¯11.6 vs. 68.4% ±â€¯4.9), cytoplasmic maturation (100.0% ±â€¯0.7 vs. 75.0% ±â€¯0.7), reactive oxygen species levels (0.5 ±â€¯0.2 vs. 0.3 ±â€¯0.1), and mitochondrial membrane potential (1.1 ±â€¯0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.


Assuntos
Adenina/análogos & derivados , Artiodáctilos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ionomicina/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Adenina/farmacologia , Animais , Artiodáctilos/embriologia , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Partenogênese/fisiologia
10.
J Assist Reprod Genet ; 37(1): 77-88, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31667700

RESUMO

PURPOSE: Oocyte in vitro maturation (IVM) is a patient-friendly reproductive technology but lower success rates than IVF have limited its uptake. Capacitation-IVM (CAPA-IVM) is an innovative new IVM system currently undergoing clinical evaluation. This study aimed to determine temporal effects of the pre-IVM phase of CAPA-IVM on cumulus function and oocyte developmental competence in mildly-stimulated mice. METHODS: Immature cumulus oocyte complexes (COCs) derived from mildly stimulated (23 h PMSG) 28-day-old mice underwent pre-IVM for 0-24 h in medium containing c-type natriuretic peptide (CNP), E2, FSH and insulin, prior to IVM (CAPA-IVM). The effect of pre-IVM duration on cumulus cell function and embryo development post-CAPA-IVM/IVF was assessed. RESULTS: Day 6 blastocyst rate increased incrementally with increasing pre-IVM duration: 40.6 ± 2.0%, 45.8 ± 1.2%, 52.2 ± 3.5%, 53.3 ± 5.9%, and 59.9 ± 2.5% for 0, 2, 6, 12, and 24 h pre-IVM, respectively (P < 0.01). DNA content/COC, a measure of cumulus cell proliferation, was significantly higher with 24 h pre-IVM group compared to 0, 2, or 6 h pre-IVM (P < 0.001). Pre-IVM for 24 h significantly increased cumulus expansion and mRNA expression of matrix genes Has2 and Tnfaip6 and Areg relative to no pre-IVM control (P < 0.01). Cumulus-oocyte gap-junctional communication (GJC) was maintained throughout 24 h pre-IVM (P < 0.0001), and GJC loss was slowed during the subsequent IVM phase, whilst meiotic resumption was accelerated (P < 0.05). Pre-IVM increased COC ATP and ADP content (P < 0.05), but not AMP, ATP/ADP, and energy charge. CONCLUSION: The pre-IVM phase of CAPA-IVM improves the quality of IVM oocytes in a temporally dependent manner and significantly influences cumulus cell function including increased cell proliferation, cumulus expansion, and prolonged cumulus-oocyte GJC.


Assuntos
Células do Cúmulo/fisiologia , Desenvolvimento Embrionário , Técnicas de Maturação in Vitro de Oócitos/métodos , Meiose , Oócitos/fisiologia , Oogênese , Animais , Células do Cúmulo/citologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia
11.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801245

RESUMO

BACKGROUND: Ovaries are sensitive to chemotherapy, which may lead to early depletion of primordial follicle reserve. One strategy for gonadal function preservation is temporary ovarian suppression with Gonadotropin Releasing Hormone agonists (GnRHa) during chemotherapy. To date, GnRHa protective mechanism of action remains not fully elucidated. METHODS: We collected 260 immature cumulus cell-oocyte complexes (COC) from 111 women < 38 years old, with a normal ovarian reserve. The COC were randomly assigned to the following groups: a) control; culture with the addition of b) GnRHa; c) cyclophosphamide; d) cyclophosphamide plus GnRHa. After in vitro treatments, RNA and proteins were extracted from oocytes and cumulus cells (CC), separately. Potential effects of drugs were evaluated on GnRH receptors, apoptosis pathways, ceramide pathway, and glutathione synthesis by quantitative PCR and, whenever possible, by Western blot. RESULTS: Cyclophosphamide triggered activation of the extrinsic pathway of apoptosis mediated by BAX in CC. The co-administration of GnRHa inhibited the apoptosis pathway in CC. According to our model, the GnRHa does not directly act on oocytes, which do not express GnRH receptors. Moreover, glutathione synthesis was decreased after GnRHa treatment both in CC and oocytes. CONCLUSION: Our data suggest that the protective mechanisms induced by GnRHa is mediated by an anti-apoptotic effect on CC.


Assuntos
Apoptose/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Receptores LHRH/genética , Proteína X Associada a bcl-2/genética , Adulto , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Ceramidas/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Ciclofosfamida/farmacologia , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Humanos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Reserva Ovariana/genética , Receptores LHRH/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Receptor fas/genética , Receptor fas/metabolismo
12.
Reprod Domest Anim ; 54 Suppl 4: 65-68, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31625245

RESUMO

The serine proteases, tissue- and urokinase-type plasminogen activators (PLAT and PLAU) and their inhibitors SERPINE1/2 are regulators of plasminogen to plasmin conversion. They are widely expressed in ovarian tissues, including granulosa and cumulus cells, and their expression is regulated by gonadotropins. The aim of this work was to assess the effect of serine protease inhibitors (aprotinin and AEBSF) and SERPINE1/2 on FSH-induced cumulus cell expansion, the production of prostaglandin E2 (PGE2) and retention of hyaluronic acid (HA) in expanding cumulus. The serine protease activity proved to be essential for the production of PGE2 and also for the retention of HA; the inhibition of plasminogen activators by SERPINE1/2 had the same effect. Collectively, these data indicate that plasmin is required for proper function of expanding cumulus cells in vitro and presumably also in vivo in the pre-ovulatory follicles.


Assuntos
Células do Cúmulo/efeitos dos fármacos , Dinoprostona/metabolismo , Oócitos/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Inibidores de Serino Proteinase/farmacologia , Serpina E2/farmacologia , Animais , Aprotinina/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Ácido Hialurônico/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Sulfonas/farmacologia , Suínos
13.
In Vivo ; 33(6): 1921-1927, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31662520

RESUMO

BACKGROUND/AIM: Cumulus cells (CCs) originate from the membrane granulosa cells and surround oocytes during follicle maturation. CCs produce high levels of hyaluronan that targets CD44, which is a major tumorigenic marker. This study aimed to investigate whether CCs have a role in cell therapy by targeting CD44 in pancreatic cancer cells. MATERIALS AND METHODS: CCs were isolated from the oocytes and incubated in a hypoxic environment. BxPC-3 pancreatic cancer cells were treated with CC conditioned media for three days. RESULTS: Conditioned media of CC cells incubated in hypoxic conditions caused a 25% reduction in the viability of BxPC-3 cells. Expression of anti-apoptotic genes was down-regulated, while that of pro-apoptotic genes was upregulated. An increased number of BxPC-3 cells exhibited increased levels of reactive oxygen species and arrested in the synthesis (S) phase of the cell cycle. CONCLUSION: CCs conditioned medium induced apoptosis of pancreatic cancer cells.


Assuntos
Células do Cúmulo/citologia , Apoptose/fisiologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Terapia Baseada em Transplante de Células e Tecidos , Células do Cúmulo/metabolismo , Regulação para Baixo/fisiologia , Perfilação da Expressão Gênica/métodos , Humanos , Receptores de Hialuronatos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Neoplasias Pancreáticas/metabolismo
14.
Biomed Pharmacother ; 118: 109350, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31545267

RESUMO

MicroRNAs (miRNAs) have attracted increasing attention for their function in oocyte in vitro maturation (IVM). In this study, we aimed to explore the functional role and underlying mechanism of miR-375 in oocyte IVM. Cumulus-oocyte complexes (COCs) were cultured in standard cell culture conditions until they reach metaphase II (MII) stage. MiR-375 overexpression or knockdown was achieved by corresponding lentiviral transduction. Levels of miR-375, disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) mRNA and progesterone receptor (PGR) mRNA were detected by qRT-PCR. Western blotting was used to assess the expression of ADAMTS1 and PGR protein. The targeted interaction between miR-375 and ADAMTS1 or PGR was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Our results demonstrate that miR-375 is downregulated, and ADAMTS1 and PGR are upregulated in cumulus cells during COC maturation. MiR-375 negatively regulates COC maturation. Moreover, ADAMTS1 and PGR are two targets of miR-375 in cumulus cells. ADAMTS1 or PGR knockdown represses COC maturation and miR-375 inhibits the expression levels of ADAMTS1 and PGR in cumulus cells. Additionally, miR-375 overexpression-mediated suppressive effect on COC maturation is abated by ADAMTS1 or PGR expression restoration. In conclusion, our study suggests that miR-375 represses oocyte IVM at least partially through targeting ADAMTS1 and PGR in cumulus cells, providing a novel insight for the involvement and underlying mechanism of miR-375 in oocyte IVM.


Assuntos
Proteína ADAMTS1/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Técnicas de Maturação in Vitro de Oócitos , MicroRNAs/metabolismo , Oócitos/metabolismo , Receptores de Progesterona/metabolismo , Animais , Sequência de Bases , Bovinos , Regulação para Baixo , MicroRNAs/genética , Regulação para Cima
15.
Am J Physiol Cell Physiol ; 317(6): C1183-C1193, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532716

RESUMO

Dual-specificity phosphatase 1 (DUSP1) is differentially expressed in cumulus cells of different physiological states, but its specific function and mechanism of action remain unclear. In this study, we explored the effects of DUSP1 expression inhibition on cell cycle progression, proliferation, apoptosis, and lactate and cholesterol levels in cumulus cells and examined reactive oxygen species levels, mitochondrial function, autophagy, and the expression of key cytokine genes. The results showed that inhibition of DUSP1 in cumulus cells caused abnormal cell cycle progression, increased cell proliferation, decreased apoptosis rates, increased cholesterol synthesis and lactic acid content, and increased cell expansion. The main reason for these effects was that inhibition of DUSP1 reduced ROS accumulation, increased glutathione level and mitochondrial membrane potential, and reduced autophagy levels in cells. These results indicate that DUSP1 limits the biological function of bovine cumulus cells under normal physiological conditions and will greatly contribute to further explorations of the physiological functions of cumulus cells and the interactions of the cumulus-oocyte complex.


Assuntos
Apoptose/genética , Ciclo Celular/genética , Células do Cúmulo/metabolismo , Fosfatase 1 de Especificidade Dupla/genética , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Autofagia/genética , Bovinos , Proliferação de Células/genética , Colesterol/metabolismo , Células do Cúmulo/citologia , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 1 de Especificidade Dupla/metabolismo , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/genética , Estresse Oxidativo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
16.
PLoS One ; 14(8): e0221663, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31442286

RESUMO

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulus-oocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats.


Assuntos
Cabras/fisiologia , Técnicas de Maturação in Vitro de Oócitos , Peptídeo Natriurético Tipo C/farmacologia , Oócitos/citologia , Animais , Antioxidantes/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Cromatina/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , DNA Mitocondrial/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Estradiol/farmacologia , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
17.
Theriogenology ; 139: 81-89, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31377650

RESUMO

The collagen type I alpha 1 chain (COL1A1), as a major component of extracellular matrix, plays a potential role in the growth and development of bovine follicles. However, its specific role in bovine cumulus cells remains unclear. In this study, we examined apoptosis, the cell cycle and reactive oxygen species after inhibition of COL1A1 expression by siRNA in bovine cumulus cells. Cell proliferation was measured by CCK-8, and mitochondrial membrane potential was detected by fluorescence intensities of JC-1 staining. Moreover, cell autophagy was detected by immunofluorescence, and cell migration was detected by a cell scratch assay. Lactic acid and cholesterol concentration were measured to evaluate the glucose utilization and cholesterol synthesis activity in cumulus cell by optical density detection method. RT-qPCR and Western blot analysis were used to measure changes in key gene expression. The results showed that cumulus cells were found to have an abnormal cell cycle, and the numbers of cells in S phase were significantly reduced, accompanied by decreases in cholesterol synthesis, and cell proliferation ability and an increase in apoptosis rate with siRNA-COL1A1 treatment. These findings were likely due to inhibition of COL1A1 resulting in high levels of ROS in the cells, a decrease in mitochondrial membrane potential, an increase in intracellular autophagy, activation of the apoptotic pathway, and a decrease in lactic acid conversion ability. COL1A1 plays an important role in regulating the physiological and biological functions of bovine cumulus cells.


Assuntos
Apoptose , Autofagia , Colágeno Tipo I/fisiologia , Células do Cúmulo/citologia , Estresse Oxidativo , Animais , Bovinos , Ciclo Celular , Proliferação de Células , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Células do Cúmulo/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo
18.
Am J Pathol ; 189(10): 2036-2045, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31369754

RESUMO

Bile acids (BAs) are present in ovarian follicular fluid (FF) and are linked to embryo development. However, information on the source of ovarian BA is scarce. Therefore, we aimed to explore local ovarian synthesis and BA transport from blood into FF. BA levels were determined in matching FF and serum from women undergoing in vitro fertilization. In vitro BA production by human mural granulosa cells (MGCs) and cumulus granulosa cells (CGCs) was measured by mass spectrometry. Gene and protein expression were quantified in MGC and CGC and in human ovarian tissue by quantitative PCR and Western blot/immunohistochemistry, respectively. BA levels in blood and FF were significantly correlated (rs = 0.186, P = 0.027) but were almost twofold higher in FF (P < 0.001). Primary BA levels were increased in FF, indicating that, in addition to passive diffusion, other sources of ovarian BA might exist. The key BA synthesis enzyme cytochrome P450 A1 was absent in MGC and CGC; BA production in vitro was undetectable. Therefore, local ovarian BA production is unlikely. However, common BA importers (Na+/taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter) and an exporter (ATP-binding cassette subfamily C member 3) were identified in GC, theca cells, and oocyte. In summary, these results suggest that passive and active transport of BAs from blood into FF constitute sources of FF BA.


Assuntos
Ácidos e Sais Biliares/metabolismo , Células do Cúmulo/metabolismo , Líquido Folicular/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Células Cultivadas , Células do Cúmulo/citologia , Feminino , Humanos , Folículo Ovariano/citologia
19.
Zygote ; 27(5): 272-278, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31411132

RESUMO

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 µM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 µm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.


Assuntos
Ácidos Nucleicos Livres/metabolismo , DNA Mitocondrial/metabolismo , Células da Granulosa/fisiologia , Compostos de Anilina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Compostos de Benzilideno/farmacologia , Sobrevivência Celular , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/genética , Células Cultivadas , Meios de Cultura/análise , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Feminino , Líquido Folicular/citologia , Líquido Folicular/fisiologia , Células da Granulosa/metabolismo , Oócitos/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Suínos
20.
Zygote ; 27(6): 382-385, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31451120

RESUMO

We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2-4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus-oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.


Assuntos
Anisóis/farmacologia , Antioxidantes/metabolismo , Blastocisto/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Bovinos , Meios de Cultura/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização In Vitro , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/metabolismo
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